Herbo-mineral formulation for the treatment of cardio vascular diseases and method of preparation thereof

ABSTRACT

Herbo-mineral Formulation for the treatment of Cardio vascular diseases and method of preparing the same are disclosed herein. The disclosed herbo-mineral formulation includes herb and mineral components which facilitate in treating Cardio vascular diseases. Cardio vascular diseases may include any condition associated with heart and blood vessels. Further, the disclosed formulation may also be instrumental as anti-oxidating, anti-stress, hypolipidemic, atherogenic, antihypertensive, apoptotsis inhibiting and cardio-protective agent.

CROSS REFERENCE TO RELATED APPLICATION

This application is based on and derives the benefit of U.S. ProvisionalApplication 62/490,213 filed on Apr. 26, 2017, the contents of which areincorporated herein by reference.

TECHNICAL FIELD

The embodiments disclosed in this specification relates to herbo-mineralformulation effective in treatment and prevention of Cardio vascular andrelated complications. It also relates to the process of preparation ofsuch formulation.

BACKGROUND

Cardiovascular diseases (CVDs) have been observed to be one of theleading causes of death globally. It is a group of diseases that areassociated with heart and blood vessels. It includes diseases such asCoronary artery disease, Cardiovascular disease, hypertensive heartdisease, peripheral arterial disease, etc.

Atherosclerosis, a condition wherein plaque builds-up in the arteries,is a leading cause of CVD. The risk factors of CVD include high bloodpressure, hypertension, stress, hyperlipidemia, Diabetes, physicalinactivity, Obesity, etc. These are the risk factors that may beregulated to prevent CVDs. There also exists other risk factors such asold age, gender, family history, etc. that cannot be regulated.

Most CVDs can be prevented by mitigating the established risk factors.Implementation of certain lifestyle modifications such as maintaining ahealthy diet, limited alcohol consumption, tobacco cessation, reducedsugar consumption, stress management, etc alone can prove helpful inmitigating the risk of developing CVDs. However, if lifestylemodifications prove to be inadequate in preventing CVD, medication ormedical procedure may be necessary.

Being one of the leading causes of death globally, extensive research inorder to develop drugs capable of treating CVD has been performed.Modern medicine offers a wide array of drugs for the same. Treatmentswith these medicines depend on the type of CVD. The various types ofdrugs include ACE inhibitors, Antiarrhythmic, Angiotensin II receptorblockers, Calcium Channel blockers, Digoxin, Diuretics, Nitrates, etc.However, managing CVDs may be a lifelong effort and may requireextensive usage of medication. Allopathic interventions often have anuntoward or undesirable side effect when used extensively.

Alternatively, ayurvedic interventions have also been known in treatingCardiovascular Diseases. Many herbal formulations have been developedbased on the knowledge of the healing properties of various herbs.However, the effectiveness of such formulations is arguable. Thereexists a need for an effective method of treating/managingCardiovascular Diseases.

OBJECTS OF THE DISCLOSED EMBODIMENTS

The principal object of the embodiments disclosed herein is to provide acomposition and method for treating Cardio vascular diseases.

A second object of the embodiments disclosed herein is to provide acomposition and method for preventing Cardio vascular diseases.

Another object of the embodiments disclosed herein is to provide aherbo-mineral formulation and a method for its preparation.

These and other objects of the embodiments herein will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following descriptions, while indicatingpreferred embodiments and numerous specific details thereof, are givenby way of illustration and not of limitation. Many changes andmodifications may be made within the scope of the embodiments hereinwithout departing from the spirit thereof, and the embodiments hereininclude all such modifications.

BRIEF DESCRIPTION OF FIGURES

The embodiments disclosed herein are illustrated in the accompanyingdrawings, throughout which like reference letters indicate correspondingparts in the various figures. The embodiments herein will be betterunderstood from the following description with reference to thedrawings, in which:

FIG. 1(a) depicts a flowchart for the preparation of Swarna MakshikaBhasma;

FIG. 1(b) depicts a flowchart for the preparation of Abhraka Bhasma;

FIG. 1(c) depicts a flowchart for the preparation of Loha Bhasma;

FIG. 1(d) depicts a flowchart for the preparation of Mukta sukti Bhasma;

FIG. 1(e) depicts a flowchart for the preparation of Pravala Bhasma;

FIG. 2 depicts a flowchart for the preparation of fortified tablets;

FIG. 3 depicts the effect of Test Drug on CK-MB activity;

FIG. 4 depicts the effect of Test Drug on Na+K+ATPase;

FIG. 5 depicts the effect of Test Drug on Mg2+ATPase;

FIG. 6 depicts the effect of Test Drug on Ca2+ATPase;

FIG. 7 depicts the effect of Test Drug on Lipid Peroxidation content;

FIG. 8 depicts the effect of Test Drug on Superoxide dismutase activity;

FIG. 9 depicts the effect of Test Drug on GSH activity;

FIG. 10 depicts the effect of Test Drug on GPX activity;

FIG. 11 depicts the effect of Test Drug on apoptotic markers;

FIG. 12 depicts the effect of Test Drug Systolic Blood pressure; and

FIG. 13 depicts the effect of Test Drug Diastolic Blood pressure;according to embodiments as disclosed herein.

DETAILED DESCRIPTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

The embodiments herein achieve a herbo-mineral formulation oftherapeutic value, and a process for the preparation of the formulation.The herbo-mineral formulation disclosed herein is useful in thetreatment and prevention of Cardiovascular diseases and complicationsrelated to cardiovascular system. In the various embodiments herein,cardiovascular Diseases may include any condition associated to heartand blood vessels such as cardiac arrhythmias, ischemic heart diseases,coronary artery disease, valve defects, etc. Further, the complicationsrelated to cardiovascular system may be any condition generally known tobe related to or considered as risk factors of CVDs includinghypertension, increased blood sugar, hyperlipidemia, etc. The disclosedformulation also find use as an anti-oxidating, anti-stress,hypolipidemic, atherogenic, antihypertensive, apoptotsis inhibiting andcardio-protective agent. Accordingly, the embodiments disclosed hereinachieve a method for the treatment/prevention of Cardiovasculardiseases. Also disclosed are embodiments of a method of reducing therisks of Cardiovascular diseases.

Formulation

The disclosed embodiments herein provide herbo-mineral formulationhaving herbs and minerals. In an embodiment, the herbo-mineralformulation includes a herb component and a mineral component. Inanother embodiment, the herbo-mineral formulation includes a herbcomponent, a mineral component and a suitable excipient.

Herb Component

In an embodiment, the herb component includes the herbs Terminaliaarjuna, Sida rombifolia, Withania somnifera, Tinospora cordifolia,Punica granatum, Embelia ribes, Rubia cordifolia, Nardostachysjatamansi, Emblica officinalis, Terminalia chebula, Terminaliabellerica, Piper longum, Piper nigrum, Zingiber officinalis, Boerhaviadiffusa, Bamboo manna, Madhuka indica, Azhadirachta indica, Picrorhizakurroa, Holy basil, Commiphora mukul, Steriospermum suaveolens, Premnamucronata, Gmelina arborea, Aegle marmelos, Oroxylum indicum, Desmodiumgangeticum, Uraria picta, Solanum indicum and Solanum xanthocarpum ortheir extracts, or the active ingredients extracted from these herbs.

In an embodiment, the herb component may include the herb as a whole ormay include specific parts of the herb such as roots, fruits, stem,leaves, rhizome, etc. In an embodiment, the herb component includes stembark of Terminalia arjuna; root of Sida rombifolia, Withania somnifera,Rubia cordifolia, Nardostachys jatamansi, Boerhavia diffusa, Picrorhizakurroa, Steriospermum suaveolens, Premna mucronata, Gmelina arborea,Aegle marmelos, Oroxylum indicum, Solanum indicum; fruit of Embeliaribes, Emblica officinalis, Terminalia chebula, Terminalia bellerica,Piper longum, Piper nigrum, Tribulus terrestris; bark of Azhadirachtaindica; plant of Solanum xanthocarpum, Uraria picta, Desmodiumgangeticum; stem of Tinospora cordifolia; fruit pericarp of Punicagranatum; rhizome of Zingiber officinalis; exudate of Bamboo manna;flower of Madhuka indica; leaves of Holy basil and gum resin ofCommiphora mukul or their extract. However, it is also within the scopeof the claims for the herb component to include other parts of the herbsuch as leaf, flowers, etc. without otherwise deterring intendedfunction of the herbo-mineral formulation.

Herbs (in whole or part), disclosed herein, maybe included in theformulation in any form that is generally known in the field. Forexample, the herbs may be processed to form extracts, dried, powdered,pelleted, concentrated, etc. In an embodiment, the herbs are dried andpowdered which is further incorporated into the formulation.

In an embodiment, the herb component includes Terminalia arjuna in anamount in the range of 8 to 12 wt %, Sida rombifolia in an amount in therange of 2 to 6 wt %, Withania somnifera in an amount in the range of 2to 6 wt %, Tinospora cordifolia in an amount in the range of 2 to 6 wt%, Punica granatum in an amount in the range of 2 to 6 wt %, Emblicaofficinalis in an amount in the range of 2 to 6 wt % and Commiphoramukul in an amount in the range of 2 to 6 wt %. Further, in anotherembodiment, the herb component includes atleast one of Embelia ribes,Rubia cordifolia, Nardostachys jatamansi, Terminalia chebula, Terminaliabellerica, Piper longum, Piper nigrum, Zingiber officinalis, Boerhaviadiffusa, Bamboo manna, Madhuka indica, Azhadirachta indica, Picrorhizakurroa, Holy basil, Steriospermum suaveolens, Premna mucronata, Gmelinaarborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Urariapicta, Solanum indicum, Solanum xanthocarpum and Tribulus terrestris,preferably than amount of ≤4 wt %.

Mineral Component

In an embodiment, the mineral component includes Bhasmas or calcinedpreparations such as Swarna Makshika bhasma, Abhraka bhasma, Lohabhasma, Muktasukti bhasma, and Pravala bhasma. Alternatively, themineral component may also be selected from a group consisting ofatleast one of Iron, Mica, Copper pyrite and Coral. In the disclosedembodiments, the bhasmas along with the herb component form bioavailableherbo-mineral complexes which are useful in treating Cardio vascularDiseases and related complications. In another embodiment, the mineralcomponent further includes Shilajit. However, it is also within thescope of claims provided herewith for the herbo-mineral formulation toinclude, as a substitute or additionally, other similar calcinedpreparations or minerals without otherwise deterring from the intendedfunction of the herbo-mineral formulation.

In an embodiment, the mineral component includes shilajit in the rangeof 1 to 4 wt %. In another embodiment, the mineral component includesMuktasukti bhasma in an amount of ≤2 wt %, Loha bhasma in an amount of≤2 wt %, Abhraka bhasma in an amount of ≤3 wt %, Swarnamaksika bhasma inan amount of ≤2 wt % and Pravala bhasma in an amount of ≤2 wt %.

The disclosed formulation, in the various embodiments herein, mayfurther include a suitable excipient. The list of suitable excipientsincludes solvents, binders, lubricants, herbal carriers, oils and saltsthat are generally known in the art. In an embodiment, the excipientincludes acacia gum.

Further, the amount of herb component and mineral component that may beincluded in the various embodiments of the disclosed formulation may bein the range of 0 to 12 wt %. In an embodiment, the formulation includesTerminalia arjuna (8 to 12 wt %), Sida rombifolia (2 to 6 wt %),Withania somnifera (2 to 6 wt %), Tinospora cordifolia (2 to 6 wt %),Punica granatum (2 to 6 wt %), Emblica officinalis (2 to 6 wt %) andCommiphora mukul (2 to 6 wt %), Shilajit 1 to 4 wt %), Muktasukti bhasma(≤2 wt %), Loha bhasma (≤2 wt %), Abhraka bhasma (≤3 wt %),Swarnamaksika bhasma (≤2 wt %) and Pravala bhasma (≤2 wt %).

In another embodiment, the formulation further includes atleast one ofEmbelia ribes, Rubia cordifolia, Nardostachys jatamansi, Terminaliachebula, Terminalia bellerica, Piper longum, Piper nigrum, Zingiberofficinalis, Boerhavia diffusa, Bamboo manna, Madhuka indica,Azhadirachta indica, Picrorhiza kurroa, Holy basil, Steriospermumsuaveolens, Premna mucronata, Gmelina arborea, Aegle marmelos, Oroxylumindicum, Desmodium gangeticum, Uraria picta, Solanum indicum, Solanumxanthocarpum and Tribulus terrestris, preferably in an amount of ≤4 wt %

Further, the amount of gum acacia may be any amount suitable to performthe activity of an excipient. In an embodiment, the formulation mayinclude gum acacia in the range of 0 to 50 mg per 500 mg of theformulation, preferably 10 wt %.

However, it is apparent that slight variations in the amount of theingredients may be performed without otherwise deterring from theintended function of the herbo-mineral formulation.

The herbo-mineral formulation disclosed herein may be formulated invarious dosage forms such that it is suitable for oral administration.The herbo-mineral formulation may be in the form of powder, tablets,pellets, lozenges, granules, capsules, solutions, emulsions,suspensions, or any other form suitable for use. In an embodiment, theherbo-mineral formulation is formulated in the form of powder suitablefor oral administration. In another embodiment, the herbo-mineralformulation is formulated in the form of tablets, preferably 500 mgtablets. For example: Table 1 depicts the quantities of each ingredientin a 500 mg tablet.

Further disclosed herein, is a tablet for treating/preventing CVDs andrelated complications. In an embodiment, the tablet is a 500 mg tablethaving herb component, mineral component and an excipient as depicted inTable 1.

TABLE 1 Each 500 mg tablet includes: BOTANICAL/ S. SCIENTIFIC QUAN- NO.SANSKRIT NAME NAME TITY 1 Arjuna dry stem bark Terminalia arjuna 50 mg 2Bala dry root Sida rombifolia 20 mg 3 Ashvagandha dry root Withaniasomnifeera 20 mg 4 Guduchi dry stem Tinospora cordifolia 20 mg 5 Dadimadry fruit pericarp Punica granatum 20 mg 6 Vidanga dry fruit Embeliaribes 10 mg 7 Manjishtha dry root Rubia cordifolia 10 mg 8 Jatamamsi dryroot Nardostachys jatamansi 10 mg 9 Amalaki dry fruits Emblicaofficinalis 20 mg 10 Hareetaki dry fruits Terminalia chebula 10 mg 11Vibhitaki dry fruits Terminalia bellerica 10 mg 12 Pippali dry fruitPiper longum 10 mg 13 Maricha dry fruit Piper nigrum 10 mg 14 Shunthidry rhizome Zingiber officinalis 10 mg 15 Punarnava dry root Boerhaviadiffusa 10 mg 16 Vamshalochana exudate Bamboo manna 10 mg 17 Madhuka dryflower Madhuka indica 10 mg 18 Nimba dry bark Azhadirachta indica 10 mg19 Katuki dry root Picrorhiza kurroa 10 mg 20 Tulasi dry leaves Holybasil 10 mg 21 Guggulu oleo gum resin Commiphora mukul 20 mg 22 Pataladry root Steriospermum suaveolens 10 mg 23 Agnimantha dry root Premnamucronata 10 mg 24 Gambhari dry root Gmelina arborea 10 mg 25 Bilva dryroot Aegle marmelos 10 mg 26 Shyonaka dry root Oroxylum indicum 10 mg 27Shalaparni dry plant Desmodium gangeticum 10 mg 28 Prshniparni dry plantUraria picta 10 mg 29 Brhati dry root Solanum indicum 10 mg 30 Kantakaridry plant Solanum xanthocarpum 10 mg 31 Gokshura dry fruit Tribulusterrestris 10 mg 32 Muktasukti bhasma Incinerated Pearle oyster 05 mg 33Loha bhasma Incinerated Iron 05 mg 34 Abhraka bhasma Incinerated Mica 10mg 35 Swarnamaksika bhasma Incinerated Copper pyrite 05 mg 36 Pravalabhasma Incinerated Coral 05 mg 37 Shilajatu fossil resin Asphaltum 10 mgExcipient Gum acacia 50 mg

Embodiments of the disclosed herbo-mineral formulation (also referred toas ‘drug’ or ‘test drug’) in tablet form were analyzed for phytoconstituents, physicochemical etc, by methods generally known in thefield. The analysis and results obtained are included hereunder asexamples by way of illustration only, and should not be construed tolimit the scope of the claims provided herewith. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of theclaims.

Example 1

Physico-chemical investigation: Physicochemical investigations like Ashvalue, tablet hardness, disintegration time, alcohol soluble extractivevalue and chloroform soluble extractive values were analyzed as per theparameters given in Indian Pharmacopeia of Ayurveda. The tabletdisintegration time was checked with the help of Tablet disintegrationmachine (I.P.Std.Rotek) and Tablet hardness tester (Secor.India) used tofind out the hardness of the tablet. Each experiment was repeatedthrice. Table 2 depicts the results of Physiochemical analysis.

TABLE 2 TEST PARAMETERS SPECIFICATIONS Description Dark brown coloredbiconvex discs Identification Positive for Iron, Calcium Average weight500 mg ± 12.5 mg Uniformity of weight ±2.5% of actual average weightTablet hardness 3.6 kg/cm² Loss on drying 6.2% w/w Methanol solubleextractive 41.2% w/v Chloroform soluble extractive 12.0% w/v Ash value15.2% w/w Average Disintegration time 26 minutes ASSAY Each tabletcontains, Iron - 4.5 mg, Calcium - 15 mg

Example 2

Phyto Constituents Study:

Tests were performed to screen the various phyto constituents such asglycosides, steroids, saponins, proteins, tannins etc.

Table 3 depicts the results of Qualitative analysis performed for phytoconstituents. The test showed the presence of alkaloids, steroids,glycosides etc which could make the drug capable of curing diseases.

TABLE 3 Aqueous Methanol Ethanol Test for Extract Extract ExtractProteins − − + Carbohydrates + + + Phenols − − − Tannins ++ ++ ++Flavonoids ++ ++ ++ Saponins + + + Terpenoids − − + Glycosides + + +Steroids − + + Alkaloids + + + (+) denotes presence and (−) denotesabsence

Method

Disclosed herein are embodiments of a method of preparing theherbo-mineral formulation. In an embodiment, the method includes:

-   -   levigating bhasmas and shilajit in a grinder;    -   adding finely powdered herbs into the grinder; and    -   adding grinding decoction while continuing grinding to obtain a        ground mass.

The bhasmas include atleast one of Muktasukti bhasma, Loha bhasma,Abhraka bhasma, Swarnamaksika bhasma and Pravala bhasma. The mixture ofbhasmas and Shilajit may be in semi solid form. In an embodiment, thelevigation may be performed for a duration of around 3 hours.

Further, the finely powdered herbs include finely powdered stem bark ofTerminalia arjuna, root of Sida rombifolia, root of Withania somnifera,root of Rubia cordifolia, root of Nardostachys jatamansi, root ofBoerhavia diffusa, root of Picrorhiza kurroa, root of Steriospermumsuaveolens, root of Premna mucronata, Gmelina arborea, root of Aeglemarmelos, root of Oroxylum indicum, root of Solanum indicum, fruit ofEmbelia ribes, fruit of Emblica officinalis, fruit of Terminaliachebula, fruit of Terminalia bellerica, fruit of Piper longum, fruit ofPiper nigrum, fruit of Tribulus terrestris, bark of Azhadirachta indica,plant of Solanum xanthocarpum, plant of Uraria picta, plant of Desmodiumgangeticum, stem of Tinospora cordifolia, fruit pericarp of Punicagranatum, rhizome of Zingiber officinalis, exudate of Bamboo manna,flower of Madhuka indica, leaves of Holy basil and gum resin ofCommiphora mukul.

The grinding decoction is a decoction of selected herbs (also referredas grinding herbs). In an embodiment, the grinding decoction is adecoction of one or more grinding herbs selected from a group consistingof: Terminalia arjuna, Asparagus racemosus, Asafetida, Cuminum cyminum,Plumbago rosea, Baliospermum montanum, Ocimum sanctum, Aloevera,Plantago ovata, Steriospermum suaveolens, Premna mucronata, Gmelinaarborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Urariapicta, Solanum indicum, Solanum xanthocarpum and Tribulus terrestris.

The decoction may be obtained by any method of decocting generally knownin the field. In an embodiment, the method of preparation of grindingdecoction includes,

-   -   soaking the grinding herbs; for example: soaking powdered dry        bark of Terminalia arjuna, fresh root of Asparagus racemosus,        resin Asafetida, dry fruit of Cuminum cyminum, dry root of        Plumbago rosea, dry root of Baliospermum montanum, dry leaves of        Ocimum sanctum, fresh leaves of Aloevera, dry seeds of Plantago        ovata, dry roots of Steriospermum suaveolens, dry roots of        Premna mucronata, dry root of Gmelina arborea, dry root of Aegle        marmelos, dry root of Oroxylum indicum, dry plant of Desmodium        gangeticum, dry plant of Uraria picta, dry root of Solanum        indicum, dry plant of Solanum xanthocarpum and dry fruit of        Tribulus terrestris, and concentrating the soaked herb mixture.

In an embodiment, soaking may be performed by soaking the grinding herbsin 16 parts of water overnight. In a further embodiment, concentratingmay be performed by boiling at high temperature, preferably about 80° C.to 85° C., until ⅛th of the liquid remains. Concentration may beconfirmed with the help of Brix meter.

Further, once the grinding decoction is added, grinding is continued. Inan embodiment, grinding is continued for about 72 hours, preferably at120 rpm, to obtain a ground mass. In an embodiment, the method ofpreparation may further include adding excipient to the ground mass,wherein gum acacia may be added to the ground mass by dissolving in thegrinding decoction, while continuing grinding for 3 hours to obtain asemisolid mass. The method of preparation may further include drying at50° C.-60° C., preferably in a hot air oven; wet granulating; andpunching to obtain 500 mg tablets. FIG. 2 depicts a flowchart for thepreparation of fortified tablets. Table 4 depicts an embodiment of theHerbs required for grinding (grinding herbs).

TABLE 4 List of grinding herbs Decoction of following herbs: 1 Arjunadry bark Terminalia arjuna 1 part 2 Shatavari fresh root Asparagusracemosus 1 part 3 Hingu resin Asafetida 1 part 4 Jeeraka dry fruitCuminum cyminum 1 part 5 Chitraka dry root Plumbago rosea 1 part 6 Dantidry root Baliospermum montanum 1 part 7 Tulasi dry leaves Ocimum sanctum1 part 8 Kumari fresh leaf Aloevera 1 part 9 Ishvaragola dry seedsPlantago ovata 1 part 10 Patala dry root Steriospermum suaveolens 1 part11 Agnimantha dry root Premna mucronata 1 part 12 Gambhari dry rootGmelina arborea 1 part 13 Bilva dry root Aegle marmelos 1 part 14Shyonaka dry root Oroxylum indicum 1 part 15 Shalaparni dry plantDesmodium gangeticum 1 part 16 Prshnipami dry plant Uraria picta 1 part17 Brhati dry root Solanum indicum 1 part 18 Kantakari dry plant Solanumxanthocarpum 1 part 19 Gokshura dry fruit Tribulus terrestris 1 partJala Water 304 parts Avashesha ⅛ part of water

The bhasmas that are used in the various embodiments of the disclosedherbo-mineral formulation may be prepared by methods that are generallyknown in the field. Bhasmas may be prepared by selecting genuinestandard minerals as starting material such as Swarnamakshika, Mica,Iron, pearl oyster etc; drying in a hot air oven; purifying the mineralby triturating, quenching, boiling, etc; triturating with herbaldecoction; preparing into discs; drying of discs; preparing sharavasamputa, subjecting Sharavasam puta to Gaja puta, and powdering of discsonce cooled. As is generally known in the field, once the disc ispowdered, the powder may again be subjected to many repetitions oftrituration with herbal decoction followed by preparing into discs;drying of discs; preparing sharavasam puta, subjecting Sharavasam putato Gaja puta, and powdering, until bhasma is obtained. The number ofrepetitions may vary from 0 to 30 times. In an embodiment, the method isrepeated as many as 30 times till bhasma is obtained.

The starting materials used in the preparation of bhasmas may includestandard minerals generally used in the field. In an embodiment, thepreparation of Swarna Makshika Bhasma includes swarna makshika as thestarting material. FIG. 1(a) depicts a flowchart for the preparation ofSwarna Makshika Bhasma using swarna makshika as the starting material.

In another embodiment, the preparation of Abhraka Bhasma includes Micaas the starting material. FIG. 1(b) depicts a flowchart for thepreparation of Abhraka Bhasma using Mica as the starting material. In anembodiment, the preparation of Loha Bhasma includes steel iron as thestarting material. FIG. 1(c) depicts a flowchart for the preparation ofLoha Bhasma using steel iron as the starting material. Further, in anembodiment, the preparation of Muktasukti Bhasma includes pearl oysteras the starting material. FIG. 1(d) depicts a flowchart for thepreparation of Muktasukti Bhasma using alloys of Pearl oyster as thestarting material. In an embodiment, the preparation of Pravala Bhasmaincludes Coral as the starting material. FIG. 1(e) depicts a flowchartfor the preparation of Pravala Bhasma using Coral as the startingmaterial.

In an embodiment, purification of mineral includes mixing the mineralwith rocksalt and lemon juice and heating strongly till partiallyoxidized into reddish powder which is further be used in the preparationof Swarna makshika Bhasma. In another embodiment, purification ofmineral includes quenching of mineral in Cow's milk wherein it isfurther used in the preparation of Abhraka Bhasma.

In yet another embodiment, purification of mineral includes quenching ofmineral in Triphala decoction which is further used in the preparationof Loha Bhasma. In yet another embodiment, purification of mineralincludes quenching of mineral in Kanjika (sour medicated gruel) which isfurther used in the preparation of Mukta sukti Bhasma.

Further, in an embodiment, purification of mineral includes boiling themineral in alkaline solution of Barilla which is further used in thepreparation of Pravala Bhasma.

The herbal decoction used may be any herbal decoction that is generallyused for triturating in the preparation of bhasmas. In an embodiment,the herbal decoction includes atleast one of Nimbu Swarasa (Lemon juice)and Kulatha Kwatha (Decoction of Dolichos biflorus), wherein it isuseful in the preparation of Swarna Makshika bhasma. In anotherembodiment, the herbal decoction includes atleast one of Arka Ksheera(Latex of calotropes procera), Snuhi Ksheera (Latex of Euphorbianeriifolia), Vata Ksheera (Latex of Ficus bengalensis), Kakamachi Rasa(fresh juice of Solanum nigrum whole plant), Gokshura Kwatha (decoctionof tribulus terrestris fruits), Apamarga Rasa (Juice of Achyranthusaspera plant), Vata Praroha Swarasa (juice of aerial root of Ficusbengalensis), Gomutra (Cow urine), Tulasi Swarasa (Fresh juice of Ocimumsanctum leaves), Kadali Shipha Jala (Juice of plantain rhizome), Erandapatra rasa (Juice of Ricinus communis leaves), and Guda (Jaggery),wherein it is useful in the preparation of Abhraka Bhasma. In anembodiment, the herbal decoction includes Triphala Kashaya (decoction offruits of Terminalia chebula, Terminalia bellerica and Emblicaofficinalis), wherein it is useful in the preparation of Loha Bhasma. Inan embodiment, the herbal decoction includes Aloe Vera juice, wherein itis useful in the preparation of Muktasukti Bhasma. In anotherembodiment, the herbal decoction includes atleast one of Aloe vera(fresh juice of leaves), Sesbania seban (fresh juice of leaves),Asparagus racemosus (fresh juice of roots) and Cow milk, wherein it isuseful in the preparation of Pravala Bhasma.

Treatment

Disclosed herein are embodiments of a method of treating/preventingCardio vascular diseases. Also disclosed are embodiments of a method ofreducing the risks of CVD.

In an embodiment, the method includes administering to a patient acomposition having a herb component, a mineral component and a suitableexcipient, wherein

-   -   the herb component includes the herbs Terminalia arjuna (8 to 12        wt %), Sida rombifolia (2 to 6 wt %), Withania somnifera (2 to 6        wt %), Tinospora cordifolia (2 to 6 wt %), Punica granatum (2 to        6 wt %), Emblica officinalis (2 to 6 wt %), Commiphora mukul (2        to 6 wt %) and atleast one herb selected from the group        consisting of Terminalia arjuna, Sida rombifolia, Withania        somnifera, Tinospora cordifolia, Punica granatum, Embelia ribes,        Rubia cordifolia, Nardostachys jatamansi, Emblica officinalis,        Terminalia chebula, Terminalia bellerica, Piper longum, Piper        nigrum, Zingiber officinalis, Boerhavia diffusa, Bamboo manna,        Madhuka indica, Azhadirachta indica, Picrorhiza kurroa, Holy        basil, Commiphora mukul, Steriospermum suaveolens, Premna        mucronata, Gmelina arborea, Aegle marmelos, Oroxylum indicum,        Desmodium gangeticum, Uraria picta, Solanum indicum and Solanum        xanthocarpum; and    -   the mineral component includes Shilajit (1 to 4 wt %), and        atleast on of Muktasukti bhasma (≤2 wt %), Loha bhasma (≤2 wt        %), Abhraka bhasma (≤3 wt %), Swarnamaksika bhasma (≤2 wt %) and        Pravala bhasma (≤2 wt %).

In an embodiment, the patient may be any individual in need of suchtreatment including ones having/suspected of having Cardiovasculardiseases. Further, the patient may also be any individualhaving/suspected of having any complication associated with heart andblood vessels. In an embodiment, Cardio vascular diseases includecardiac arrhythmias, ischemic heart diseases, coronary artery disease,valve defects, mitral valve prolapse etc. Further, the patient may alsobe any individual prone to or having risks of Cardiovascular diseases,for example: individuals having hypertension, obesity, increased bloodsugar, etc.

In an embodiment, the method of treating/preventing CVD includesadministering the Disclosed formulation to a patient wherein thedisclosed formulation acts as atleast one of anti-oxidating,anti-stress, hypolipidemic, atherogenic, antihypertensive, apoptotsisinhibiting and cardio-protective agent.

The disclosed method of treatment may be used as a primary line oftreatment or as an adjunct to other CVD treatment methods. In anembodiment, the method may be instrumental in improving the healthconditions of individuals having CVD.

The dosage of the test drug and the treatment regimen may vary dependingon the patient. The disclosed formulation was subjected to acute oraltoxicity study, and a study to check its effect on behavior and nervoussystem. The studies proved that Test drug is free from toxicity even ata dose of 6000 mg/kg weight which was the maximum possible dose. It wasalso found to have no harmful effects on behavior and nervous system.

The Disclosed formulation (also referred as Test drug or Test product)was further evaluated for efficacy of cardio-protective activity bypreclinical and clinical studies, as described hereunder by way ofexamples. Embodiments of the formulation disclosed herein is furtherdescribed by reference to the following examples by way of illustrationonly, and should not be construed to limit the scope of the embodimentsherein. It will be apparent to those skilled in the art that manymodifications, both to materials and methods, may be practiced withoutdeparting from the scope of the claims.

Example 3: Preclinical Study

The aim of this study was to analyze the Cardioprotective activity oftest product on Isoproterenol (ISP) induced experimental model ofmyocardial infarction. The effect of Test drug on the membrane boundenzymes like Na+K+ATPase, Ca2+ATPase and Mg2+ATPase were investigated.The action of Test drug on apoptotic and pre-apoptotic markers geneexpression in ISP induced myocardial infarction was analyzed. Also, theeffect of Test drug on CK-MB activity and oxidative stress markers wasevaluated.

Experiment Details:

Animals:

35 male Sprague—Dawley rats of 150-200 g body weight were selected forthe study. Animals were housed in individual polycarbonate cages in awell-ventilated room under an ambient temperature of 23±2 degree C. and40-65% relative humidity, with artificial photoperiod 12-h light/12-hdark cycle. They were provided with standard rodent pellet diet(Nutrilab Rodent, Tetragon Chemie, India) and purified water ad libitum(RIOS, USA). Experimental animals were acclimatized for 7 days to thelaboratory conditions prior to experimentation. The study protocol wasapproved by Institutional Animal Ethical Committee (IAEC).

Experimental Groups and Design:

Rats were randomized into 5 groups based on the body weight. ISP (120mg/kg) was injected subcutaneously to rats on 19th and 21st day toinduce experimental myocardial infarction. Test drug was orallyadministered throughout the study.

Group I (Normal control): 0.5% CMCGroup II (Positive control): 0.5% CMC+ISP (120 mg/kg)Group III (Standard): Carvedilol (2 mg/kg/day, p.o)+ISP (120 mg/kg,s.c.)Group IV (Low dose): Test drug (50 mg/kg/day, p.o)+ISP (120 mg/kg, s.c.)Group V (High dose): Test drug (100 mg/kg/day, p.o)+ISP (120 mg/kg,s.c.)

The change in body weight was recorded every week. At the end of theexperiment, blood was collected and the plasma was separated forbiochemical investigation. The animals were euthanized and the organs(heart, Kidney and adrenals) were dissected out and weighed. Hearthomogenate was used for further analysis and LV was separated for theanalysis of apoptotic marker gene expression as depicted in the variousexamples hereunder.

Biochemical Parameters Example 3(a): CK-MB (Creatinine Kinase-MB)

The CK-MB activity was measured by kit method (Spinreact, Spain) usingSemi-automated biochemical analyzer.

Effect of Test Drug on CK-MB Activity:

FIG. 3 depicts CK-MB activity. Rats induced with ISP showed significantelevation (P<0.01) in CK-MB activity when compared to normal rats,whereas pre-treatment with test drug decreased (P<0.01) its activity.

The serum marker enzyme CK-MB in ISP induced rats serves as the index toassess the severity of myocardial injury. This increase in activity isaccompanied by their concomitant increase in wet weight of myocardiumconfirms the onset of apoptotic pathway. Extent of cardio-protectionoffered by the drug is associated with the significant attenuation ofCK-MB activity. Hence, pretreatment with Test Drug significantly possesscardio-protective effect and maintains myocardial membrane integrity.

Example 3(b): Na+K+ATPase

Reagents

-   -   1. Tris HCl (184 mM) pH-7.5: 1.449 g in 50 ml of distilled water    -   2. KCl (50 mM): 37.275 mg in 10 ml of distilled water    -   3. Sodium EDTA (1 mM): 4 mg in 10 ml of distilled water    -   4. NaCl (600 mM): 350.64 mg in 10 ml. of distilled water    -   5.10% TCA: 10 g in 100 ml of distilled water    -   6.15% sodium meta bisulphate: 7.5 g in 50 ml of distilled water    -   7.20% sodium sulphate: 2 g in 10 ml of distilled water    -   8.5N H2SO4: 13.5 ml of H2SO4 in 100 ml of distilled water    -   9. Ammonium molybdate (2.5%): 2.5 g in 100 ml of 5N Sulphuric        acid    -   10. ANSA (0.1%): 100 mg in 39 ml of 15% sodium meta bisulphate.        Then 1 ml of 20% sodium sulphite was added and the volume was        made upto 100 ml with distilled water.

Protocol:

Na+K+ATPase was assayed by taking 250 μl of tris HCl buffer followed bythe addition of 50 μl of 600 mM NaCl, 50 μl of 50 mM KCL, along with 50μl of 1 mM Na. EDTA, and 50 μl of 80 mM ATP. The reaction mixture waspre-incubated at 37° C. for 10 mins. Then 25 μl of 10% homogenate wasadded to the test alone and further incubated at 37° C. for 1 hr. Thereaction was immediately arrested by the addition of 10% TCA. Thecontrol reaction rate was correspondingly assessed by adding 25 μl of10% homogenate only after arresting the reaction. The precipitate wasremoved by centrifugation at 3500 rpm for 10 minutes. To 50 μl of thesupernatant, 1075 μl of distilled water, 125 μl of Ammonium molybdateand 50 μl of ANSA were added and incubated for 10 mins at 37° C. Theintensity of blue colour was read at 640 nm using spectrophotometeragainst a blank that contained all the reagents minus the supernatant.The results are expressed in μg of Pi liberated/min/mg of protein.

Effect of Test Drug on Membrane Bound Enzymes: Na⁺ K⁺ ATPase:

FIG. 4 depicts Na⁺ K⁺ ATPase activity. It was observed that ISP inducedrats showed significant (P<0.01) decrease in Na⁺ K⁺ ATPase. Whereas,pretreatment with Test drug (50 and 100 mg/kg) showed significant(P<0.01) elevation in Na⁺ K⁺ ATPase activity irrespective of the doses.

Na⁺ K⁺ ATPase, a membrane bound enzyme is responsible for sodium ioninflux and potassium ion reflex during muscle contraction andrelaxation. Induction of myocardial infarction with ISP inhibited theinflux of sodium ions thereby hindering the contraction and relaxationof the heart muscle. Pretreatment with Test drug was comparable with thereference drug, carvedilol in enhancing the Na⁺ K⁺ ATPase activity

Example 3(c): Mg2+ATPase

Reagents:

-   -   1. Tris HCl (0.1M) pH-7.4: 1.576 g in 100 ml of distilled water    -   2. KCl (0.1M): 74.55 mg in 10 ml of distilled water    -   3. MgCl2 (0.1M): 200 mg in 10 ml of distilled water    -   4. ATP (80 mM): 440 mg in 10 ml of distilled water    -   5. 10% TCA: 10 g in 100 ml of distilled water    -   6. 15% sodium meta bisulphate: 7.5 g in 50 ml of distilled water    -   7. 20% sodium sulphate: 2 g in 10 ml of distilled water    -   8. 5N H2SO4: 13.5 ml of H2SO4 in 100 ml of distilled water    -   9. Ammonium molybdate (2.5%): 2.5 g in 100 ml of 5N Sulphuric        acid.    -   10. ANSA (0.1%): 100 mg in 39 ml of 15% sodium meta bisulphate.        Then 1 ml of 20% sodium sulphite was added and the volume was        made upto 100 ml with distilled water.

Protocol:

Total ATPase was assayed by taking 0.75 ml of tris HCL buffer followedby the addition of 50 μl of 100 mM KCl, along with 50 μl of 100 mMMgCl₂, and 50 μl of 80 mM ATP. The reaction mixture was pre-incubated at37° C. for 2 mins. Then 50 μl of 10% homogenate was added to the testalone and further incubated at 37° C. for 20 mins. The reaction wasimmediately arrested by the addition of 500 μl of 10% TCA. Controlreaction rate was correspondingly assessed by adding 50 μl of 10%homogenate only after arresting the reaction. The precipitate wasremoved by centrifugation at 3500 rpm for 10 minutes. To 50 μl of thesupernatant, 1075 μl of distilled water, 125 μl of Ammonium molybdateand 50 μl of ANSA were added and incubated for 10 mins at 37° C. Theintensity of blue colour was read at 640 nm using spectrophotometeragainst a blank that contained all the reagents minus the supernatant.The results are expressed in μg of Pi liberated/min/mg of protein.

Effect of Test Drug on Membrane Bound Enzymes: Mg²⁺ ATPase:

FIG. 5 depicts Mg²⁺ ATPase activity. It was observed that ISP inducedrats showed significant (P<0.01) decrease in Mg²⁺ ATPase activity whencompared to the normal control rats. Whereas, pretreatment with Testdrug (50 and 100 mg/kg) showed significant (P<0.01) elevation in Mg²⁺ATPase activity irrespective of the doses. Mg²⁺ ATPase regulates theintracellular Mg2+ levels. Pretreatment with Test drug was comparablewith the reference drug, carvedilol in enhancing the Mg²⁺ ATPaseactivity

Example 3(d): Ca2+ ATPase

Reagents:

-   -   1. Tris HCl (0.1M) pH-7.4: 1.576 g in 100 ml of distilled water    -   2. KCl (0.1M): 74.55 mg in 10 ml of distilled water    -   3. CaCl2 (0.1M): 147.02 mg in 10 ml of distilled water    -   4. ATP (80 mM): 440 mg in 10 ml of distilled water    -   5. 10% TCA: 10 g in 100 ml of distilled water    -   6. 15% sodium meta bisulphate: 7.5 g in 50 ml of distilled water    -   7. 20% sodium sulphate: 2 g in 10 ml of distilled water    -   8. 5N H2SO4: 13.5 ml of H2SO4 in 100 ml of distilled water.    -   9. Ammonium molybdate (2.5%): 2.5 g in 100 ml of 5N Sulphuric        acid.    -   10. ANSA (0.1%): 100 mg in 39 ml of 15% sodium meta bisulphate.        Then 1 ml of 20% sodium sulphite was added and the volume was        made upto 100 ml with distilled water.

Protocol:

Ca²⁺ ATPase was assayed by taking 0.75 ml of tris HCL buffer followed bythe addition of 50 μl of 100 mM KCl, 50 μl of 100 mM CaCl₂ and 50 μl of80 mM ATP. The reaction mixture was pre-incubated at 37° C. for 2 mins.Then 50 μl of 10% homogenate was added to the test alone and furtherincubated at 37° C. for 20 mins. The reaction was immediately arrestedby the addition of 500 μl of 10% TCA. Control reaction rate wascorrespondingly assessed by adding 50 μl of 10% homogenate only afterarresting the reaction. The precipitate was removed by centrifugation at3500 rpm for 10 minutes. To 50 μl of the supernatant, 1075 μl ofdistilled water, 125 μl of Ammonium molybdate and 50 μl of ANSA wereadded and incubated for 10 mins at 37° C. The intensity of blue colourwas read at 640 nm using spectrophotometer against a blank thatcontained all the reagents minus the supernatant. The results areexpressed in μg of Pi liberated/min/mg of protein.

Effect of Test Drug on Ca2+ATPase:

FIG. 6 depicts Ca²⁺ ATPase activity. It was observed that in ratsadministered with ISP, Ca²⁺ ATPase activity was increased when comparedto normal control rats. On the other hand, pretreatment with Test drugshowed reduced Ca²⁺ ATPase activity and the result was independent ofthe dose concentration.

Intracellular calcium (Ca²⁺) levels are maintained and regulated by Ca²⁺ATPase. Enhanced Ca²⁺ ATPase and intracellular Ca²⁺ overload inISP-treated rats can be correlated to the action of adenylate cyclase.Phosphorylation of Ca²⁺ channel protein by cAMP is expected to increasethe Ca²⁺ influx into the myocardium and thus burdening it. In our study,pre-supplementation with Test drug showed potent resistance to theperturbations in Ca²⁺ ATPase caused due to ISP injection.

Oxidative Stress—Anti-Oxidant Potential of Test Drug on ISP InducedMyocardial Infarction Example 3(e): Lipid Hydroperoxide (LPO)

Reagents:

-   -   1. ThioBarbituric Acid (TBA) (0.8%): 0.8 gms in 0.5N HCl    -   2. Butylated Hydroxyl Toluene (0.05%): 0.05 gms in methanol.    -   3. Saline (0.9%): 0.9 g in 100 ml distilled water

Protocol:

The method involved heating of 0.5 ml of heart homogenate ofexperimental rats with 0.8 ml saline, 0.5 ml of BHT and 3.5 ml TBAreagent for 11/2 min in a boiling water bath. After cooling, thesolution was centrifuged at 2,000 rpm for 10 min and the precipitateobtained was removed. The absorbance of the supernatant was determinedat 532 nm using spectrophotometer against a blank that contained all thereagents minus the biological sample. The values were expressed in mg/gtissue (Okhawa H et al., 1979).

Lipid Peroxidation (Thiobarbituric Acid Reactive Substances):

FIG. 7 depicts LPO Content. ISP induced rats showed significant increasein the levels of heart TBARS when compared to normal control rats.Pretreatment with Test drug showed considerable decrease in the levelsof heart TBARS in ISP-induced rats. The results were comparable withthat of standard drug, Carvedilol.

ISP treatment is known to produce free radical moieties via its quininemetabolites that react with oxygen ultimately resulting in enhancedgeneration of Reactive oxygen species (ROS). ROS, the highly toxicby-products of aerobic metabolism are known to react extensively withcell membranes and macromolecules enhancing formation of lipid peroxidesthus leading to tissue damage. Lipid peroxidation is an importantpathogenic event in myocardial necrosis and accumulation of lipidhydroperoxides reflects damage of the cardiac constituents. Theincreased levels of MDA, a lipid peroxidation end-product, observed inour study following isoproterenol administration might be due to freeradical mediated membrane damage. Our findings suggest that TEST DRUGpossess lipid peroxidation inhibitory activity.

Example 3(f): Superoxide Dismutase (SOD)

Reagents:

-   -   1. Sodium pyrophosphate buffer (0.025M): 1.115 g in 100 ml of        distilled water.    -   2. Phenazonium Metho Sulphate (PMS) (186 μM): 3 mg in 10 ml of    -   distilled water (930 μM). Then 1:5 dilutions were carried out to        obtain 186 μM.    -   3. Nitro Blue Tetrazolium (chloride) (NBT) (300 μM): 3 mg in 10        ml of phosphate buffer.    -   4. NADH (780 μM): 6 mg in 10 ml of phosphate buffer.

Protocol:

Superoxide dismutase was assayed by taking 0.05 ml of heart homogenatefollowed by addition of 0.3 ml of sodium pyrophosphate buffer (0.025M,PH 8.3), 0.025 ml of PMS (186 μM) and 0.075 ml of NBT (300 μM in bufferof PH 8.3) The reaction was started by addition of 0.075 ml of NADH (780μM in buffer of PH 8.3). After incubation at 300 C for 90 seconds, thereaction was stopped by addition of 0.25 ml glacial acetic acid. Thenthe reaction mixture was stirred vigorously and shaken with 2.0 ml ofn-Butanol. The mixture was allowed to stand for 10 minutes andcentrifuged. 1.5 ml of n-butanol alone was served as blank. The colourintensity of the chromogen was read at 560 nm (Kakkar, P., et al 1984).

Superoxide dismutase activity: FIG. 8 depicts SOD activity. IT wasobserved that Superoxide dismutase levels were decreased in ISP inducedrats when compared to the normal control rats. Pretreatment with TestDrug exhibited a dose dependent increase in SOD levels when compared tovehicle treated ISP induced rats. The SOD levels of Test drug treatedwere comparable with that of the standard drug, Carvedilol treated rats.

Superoxide anions and other reactive oxygen species produce an oxidativeenvironment commonly called as oxidative stress in rat myocardium. Amongthe free radical scavenging antioxidants SOD was considered to be thecellular defense against oxidative injury. The results envisaged thatthe decreased levels of SOD following ISP administration wereameliorated to a greater extend by pre-treatment with Test drug.

Example 3(g): GSH and Glutathione Peroxidase Activity

Glutathione Peroxidase [GPX]

Reagents:

-   -   1. Sodium Azide (10 mM): 16 mg in 25 ml of distilled water    -   2. GSH (2 mM): 30.732 mg in 50 ml of distilled water.    -   3. H2O2 (1 mM): 29 μl in 1000 ml of distilled water    -   4. 10% TCA: 10 g in 100 ml of distilled water    -   5. K.EDTA (0.4 mM): 16 mg in 100 ml of distilled water    -   6. Tris HCL Buffer (0.4 mM): 6.304 g in 100 ml of distilled        water    -   7. DTNB (0.6 mM): 12 mg in 50 ml PO4 buffer.

Protocol:

Glutathione peroxidase (GPX) was assayed by taking 200 μl of tris HCLbuffer (0.4 M), 0.4 mM K.EDTA along with 100 μl of sodium azide and 200μl of enzyme preparation (hemolysate) and mixed well. Thereafter, 200 μlof reduced glutathione solution (2 mM) followed by 0.1 ml H2O2 wereadded The overall reaction was arrested by adding 0.5 ml of 10% TCA. Theprecipitate was removed by centrifugation at 4000 rpm for 10 minutes.The absorbance was read at 412 nm using spectrophotometer. Thenon-enzymatic reaction rate was correspondingly assessed by replacingthe enzyme sample by buffer. The results are expressed as mg of GSHconsumed/min/mg protein (Rotruck, J et al., 1973).

Reduced Glutathione [GSH]

Reagents:

-   -   1. 5% TCA: 5 g in 100 ml of distilled water.    -   2. Phosphate buffer (pH: 8) 0.2M    -   3. DTNB (0.6 mM): 12 mg in 50 ml PO4 buffer.

Protocol:

Glutathione content was estimated according to the method (Moren et al1979). 0.25 ml of serum was added to equal volume of ice cold 5% TCA.The precipitate was removed by centrifugation at 4000 rpm for 10minutes. To 1 ml aliquot of supernatant, 0.25 ml of 0.2M phosphatebuffer, pH 8.0 and 0.5 ml of DTNB (0.6 mM in 0.2M phosphate buffer, pH8.0) was added and mixed well. The absorbance was read at 412 nm usingspectrophotometer. The values were expressed in mg/g tissue.

GSH and Glutathione Peroxidase Activity:

FIG. 9 depicts GSH activity and FIG. 10 depicts GPX activity in theheart of normal and ISP induced rats. ISP induced in rats myocardialinfarction showed significantly (p<0.01) decreased in anti-oxidantslevel of positive control group and significantly increased in GSH, GPXactivities as compared to normal control group. Pretreatment with testdrug dose dependently to ISP induced rats significantly increased theactivities of these enzymes compared with positive control group.Glutathione is known to protect the myocardium against the free radicalsmediated injury by the reduction of hydrogen peroxide, leads to decreasethe reduced glutathione levels during the induction of cardiac necrosis(Kocak, H., et al., 1992). Depressed GSH levels with enhanced protectivemechanism to oxidative stress in myocardial infarction. ISPadministration was found to reduce the level of GSH in plasma andcardiac tissue (Ji et al., 1988). GPX and GST activities aresignificantly depressed in ISP induced rats. Endogenous enzymesinactivation of GPX in the heart leads to accumulation of oxidizedglutathione (Ferrari R et al 1985). Inactivation of oxidized glutathioneby enzymes containing sulphydryl group inhibit the protein synthesis (Jiet al 1988). In present study dose dependently increased in theactivities of GSH, GPX on pretreatment with Test drug in ISP inducedmyocardial infarction group showed the antioxidant potential of Testdrug against myocyte injury caused by free radicals.

Example 3(i): Total Protein by Biuret Method

Reagents:

-   -   1. 0.2N sodium hydroxide    -   2. 0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium        tartrate.    -   3. Stock Biuret solution: Dissolve 4.5 g of sodium potassium        tartarate in 40 ml of 0.2N NaOH. Then add 1.5 g of CuSO4 until        completely dissolved. Finally add 500 mg of KI and make up the        volume to 100 ml with 0.2N NaoH.    -   4. Working Biuret solution: From stock biuret solution 1:5        dilution.    -   5. Stock Protein solution: Weigh accurately 50 mg of bovine        serum albumin (fraction V) and dissolve in distilled water and        make up to 50 ml in standard flask.    -   6. Working standard: Dilute 10 ml of the stock solution to 50 ml        with distilled water in a standard flask. One ml of this        solution contains 200 μg protein.

Protocol:

Estimation of protein was assayed by taking 0.2 ml of saline, 10%homogenate, followed by the addition of 1.25 ml of working biuretreagent. It was incubated at room temperature for 15 minutes. The colorintensity was read at 540 nm.

Isoproterenol (ISP) injected rats (positive control) showed asignificant decrease (P<0.001) in total protein levels as compared tocontrol rats. Administration of test drug to ISP injected rats (Lowdose) showed a significant (P<0.05) increase in the total protein leveland highly significant increase in protein level (almost to normallevel) is observed in both test drug group with high dose and standards(Carvedilol-2 mg/kg)

A decrease in the level of serum total proteins in Isoproterenolinjected rats could be due to increased free radical production byIsoproterenol. Administration of test drug showed dose dependentimprovement in serum protein levels as compared to ISP injected rats.This improvement is comparable to that of standard. Table 5 illustratesthe effect of test drug on Total protein.

Table 5 Anti-Apoptosis (Gene Expression) Example 3(j): ReverseTranscriptase (RT)

Group Treatment Total protein ( mg/dl) I Normal Control 7.34 ± 0.37 IIPositive Control 3.58 ± 0.65 III Standard (Carvedilol- 2 mg/kg) 7.24 ±0.32 IV Low dose (50 mg/kg) 6.89 ± 0.36 V High dose (100 mg/kg) 7.31 ±0.42

PCR was performed to determine the level of mRNA expression of Caspase,Bax, BCl2 and p53. Briefly, total RNA was extracted from left ventricle(Heart) using TRIzol Reagent (Sigma, USA). After homogenization, thetubes were incubated for 10 minutes and centrifuged at 1000 rpm for 5min. 200 μl of chloroform was added to the supernatant, allowed toincubate for 5 min at room temperature and centrifuged at 12000 rcf for20 min. Then 500 μl of isopropyl alcohol was added to the supernatant toprecipitate the total RNA and centrifuged at 12000 rcf for 15 minfollowing the incubation period of 10 min. The supernatant was decantedcarefully; the pellet was washed three times with 75% ethanol,centrifuged at 12000 rcf for 15 min and the pellet was allowed to airdry. The pellet was resuspended in 20 μl of RNase free water and storedin −80° C. until use. The isolated RNA was allowed to undergo reversetranscription and polymerization reaction to get cDNA using PCR mastercycler gradient. The formed cDNA was loaded in agarose gel, allowed torun the electrophoresis at 80V for 30 min and the gene expression wasanalyzed using the bands formed. 200 nanograms of RNA were used forreverse transcription polymerase chain reaction (RT-PCR) according tothe manufacturer's instructions (Genet Bio, Korea). The followingsequence was performed for each PCR reaction: 42.0 for 30 s, 94.0 for 5min (1 cycle); 94° C. for 1 min, Caspase (56.0), Bax (58.8), Bcl2 (56.7)and p53 (57.9) for 1 min, and 72° C. for 1 min (with 35 cycles); and afinal extension phase at 74° C. for 10 min.

The gene sequences (5′-3′) for the proteins are as follows,

Bax- Forward primer- GAGTGTCTCCGGCGAATTG Reverse primer-TGGTGAGCGAGGCGGTGAC Bcl2- Forward primer- CGGGAGATCGTGATGAAGTReverse primer- CCACCGAACTCAAAGAAGG Caspase 3- Forward primer-CTGGACTGCGGTATTGAG Reverse primer- GGGTGCGGTAGAGTAAG P53-Forward primer- GGATGCCCGTGCTGCCGAGGAG Reverse primer-AGTGAAGGGACTAGCATTGTC

Data Analysis

Statistical analysis was performed using GraphPad Prism, 4.03 (SanDiego, US). Data were expressed as mean±SEM. Mean difference wasanalyzed by one way ANOVA with Tukey's multiple comparison as the posthoc test. Probability value less than 0.05 was fixed as the statisticalsignificance criterion.

Effect of Test Drug on Apoptotic Markers:

FIG. 11 illustrates the gene expression of apoptotic marker. Inductionof myocardial infarction with ISP up-regulated the expression ofapoptotic markers, Caspase 3, P53 and Bax and down regulated theexpression of anti-apoptotic marker BCL-2. In the cascade of apoptosis,increased expression of Caspase 3 results in the phosphorylation ofother caspases, on the other hand, P53 inhibits BCL-2 expression andconversely enhances Bax expression. Bax in turn inhibits the CytochromeC thereby augmenting the reactive oxygen species generation. BCL-2 andBax proteins are known to modulate the cell survival signals of variousapoptotic stimuli (Sutton, et al., 1997). Henceforth, we investigatedthe possible outcomes of Test drug treatment in ISP induced rats.Treatment with Test drug markedly up-regulated the expression of BCL-2,whereas down-regulated the expression of Caspase-3, P53 and Bax.

Clinical Observations:

No mortality was observed between the groups. The rats that were inducedwith isoproterenol showed increased heart rates and heart palpitation.Rigidity of the muscles was observed in the animals and there wasdecreased movement and activity in the animals. Exophthalmus, theprotrusion of the eye balls, was very evident after the induction ofisoproterenol. They exhibited increased respiration rate. Further, thesymptoms of myocardial infarction were clearly visible in the ISPinjected animals and the offset of these clinical signs prolonged inpositive control.

Effect of Test Drug on Body Weight:

Table 6 depicts the body weight of animals for three weeks. Nosignificant body weight changes were observed between the experimentalgroups on day 0, 7 and 14, whereas there was significant alteration inbody weight on day 21.

TABLE 6 Body weight of animals for three weeks. Body weight (g) GroupTreatment 0^(th) day 7^(th) day 14^(th) day 21^(st) day I Normal Control201.29 ± 2.00 215.86 ± 2.50 223.43 ± 1.62 226.14 ± 3.19 II PositiveControl 206.86 ± 1.11 202.00 ± 1.19 202.71 ± 3.48 192.29 ± 1.44^(##) IIIStandard 211.00 ± 1.70 210.57 ± 3.22 218.43 ± 4.37 216.43 ± 1.58**(Carvedilol- 2 mg/kg) IV Low dose 204.71 ± 6.04 207.57 ± 5.84 216.14 ±2.97 203.29 ± 0.14** (50 mg/kg) V High dose 202.00 ± 4.99 203.71 ± 4.37207.14 ± 1.47 200.71 ± 0.39* (100 mg/kg) Values were expressed in Mean ±SEM (n = 6); ^(#), ^(##)denotes P < 0.05 and 0.01 respectively(Comparison between Normal and Positive control); *, **denotes P < 0.05and 0.01 respectively (Comparison between Positive control and othertreatment groups).

Effect of Test Drug on Organ Weight:

Table 7 depicts the organ weight of various groups. No significantdifference in kidney and adrenals weight was observed between thetreatment groups. The ISP induced MI rats showed increase in heartweight (24%) when compared to normal rats, whereas treatment with TESTDRUG altered the changes.

TABLE 7 Organ weight of various groups Organ weight (g) Group TreatmentKidney Adrenals Heart I Normal Control 0.85 ± 0.02 0.02 ± 0.01 0.41 ±0.02 II Positive Control 0.77 ± 0.06 0.02 ± 0.00 0.51 ± 0.02 (24%) IIIStandard (Carvedilol-2 mg/kg) 0.68 ± 0.10 0.02 ± 0.00 0.44 ± 0.07(13.1%) IV Low dose (50 mg/kg) 0.79 ± 0.02 0.02 ± 0.00 0.51 ± 0.02(0.2%) V High dose (100 mg/kg) 0.69 ± 0.09 0.02 ± 0.00 0.45 ± 0.06)(12.3%) Values were expressed in Mean ± SEM (n = 6); Normal control wascompared with positive and Positive control compared with othertreatment groups.

Example 4: Clinical Study

150 hypertensive patients comprising 86 men and 74 women who werealready on anti-hypertensives were advised to take Test drug along withtheir existing allopathic medicines. They were studied for a period ofsix weeks.

Dose:

Two tablets (500 mg×02) were administered twice daily swallowed withwater after food.

Results and Observation:

Table 8 depicts Mean B.P before and after 3 and 6 weeks of Test drugadministration (n=150).

TABLE 8 Mean systolic B.P Mean diastolic B.P Duration Week 0 Week 3 Week6 Week 0 Week 3 Week 6 MEAN 167.12 131.2 125.12 104.76 84.16 81 S.D.19.61 15.6 11.62 9.5 6.99 5.05 S.E.M ±0.71 ±2.77 ±1.64 ±11.34 ±0.98±0.71

FIG. 12 illustrates the effect of Test drug on Systolic B.P. and FIG. 13illustrates the effect of Test drug on Diastolic B.P.

After using the test drug, the resting pulse rates and blood pressure,both systolic and diastolic showed a significant reduction. In additionto helping in lowering of blood pressure and reduction of dosage ofallopathic drugs there was also a feeling of general well-being. Afterthe treatment with test drug for 6 months, there was an effectivereduction in the dosages of the other anti-hypertensive drug in manycases.

Note: In a case of mitral valve prolapse with mild mitral regurgitation,when the test product was administered for 8 months follow up 2DEchocardiography confirmed that mitral valve prolapse is completelyresolved and no mitral regurgitation was observed. Patient becameasymptomatic, cardiac chambers and output were within normal limits.

The aforementioned studies proved that test product is free fromtoxicity. Preclinical study shows that test product exerts itsCardioprotective action through anti-apoptotic pathway in Isoproterenolinduced experimental model of myocardial infarction. Clinical studysupports antihypertensive, cardioprotective and valve correcting actionsof the Test drug.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein can bepracticed with modification within the spirit and scope of theembodiments as described herein.

We claim:
 1. A formulation for treatment and management ofCardiovascular ailments comprising: a herb component comprisingTerminalia arjuna, Sida rombifolia, Withania somnifera, Tinosporacordifolia, Punica granatum, Emblica officinalis and Commiphora mukul,or their extracts thereof; and a mineral component comprising shilajitand bhasma.
 2. The formulation claimed in claim 1, wherein Terminaliaarjuna is present in an amount ranging from 8 to 12 wt %, Sidarombifolia is present in an amount ranging from 2 to 6 wt %, Withaniasomnifera is present in an amount ranging from 2 to 6 wt %, Tinosporacordifolia is present in an amount ranging from 2 to 6 wt %, Punicagranatum is present in an amount ranging from 2 to 6 wt %, Emblicaofficinalis is present in an amount ranging from 2 to 6 wt % andCommiphora mukul is present in an amount ranging from 2 to 6 wt %. 3.The formulation claimed in claim 1 wherein said herb component furthercomprises of atleast one herb selected from a group consisting ofEmbelia ribes, Rubia cordifolia, Nardostachys jatamansi, Terminaliachebula, Terminalia bellerica, Piper longum, Piper nigrum, Zingiberofficinalis, Boerhavia diffusa, Bamboo manna, Madhuka indica,Azhadirachta indica, Picrorhiza kurroa, Holy basil, Steriospermumsuaveolens, Premna mucronata, Gmelina arborea, Aegle marmelos, Oroxylumindicum, Desmodium gangeticum, Uraria pieta, Solanum indicum, Solanumxanthocarpum, and Tribulus terrestris; or their extracts thereof.
 4. Theformulation as claimed in claim 3, wherein said herb is present in anamount of ≤4 wt %.
 5. The formulation as claimed in claim 1, whereinsaid mineral component comprises of atleast one bhasma selected from agroup consisting of Muktasukti bhasma, Loha bhasma, Abhraka bhasma,Swarnamaksika bhasma and Pravala bhasma.
 6. The formulation as claimedin claim 1, wherein shilajit is present in an amount ranging from 1 to 3wt %.
 7. The formulation as claimed in claim 5, wherein Muktasuktibhasma is present in an amount of ≤2 wt %, Loha bhasma is present in anamount of ≤2 wt %, Abhraka bhasma is present in an amount of ≤3 wt %,Swarnamaksika bhasma is present in an amount of ≤2 wt % and Pravalabhasma is present in an amount of ≤2 wt %.
 8. The formulation as claimedin claim 1, further comprising a suitable excipient, preferably gumacacia.
 9. The formulation as claimed in claim 1, wherein saidformulation is in the form of powder.
 10. The formulation as claimed inclaim 1, wherein said formulation is in the form of tablet.
 11. Theformulation as claimed in claim 10, wherein said tablet is in the formof 500 mg tablets.
 12. Use of the formulation as claimed in claim 1 inthe preparation of a medicament for treating and preventingCardiovascular diseases.
 13. Use of the formulation as claimed in claim1 in the preparation of a medicament for reducing the risks ofCardiovascular diseases.
 14. A process for the preparation offormulation claimed in claim 1, comprising: levigating bhasmas andshilajit; adding finely powdered herbs; and adding grinding decoctionwhile continuing grinding to obtain a ground mass.
 15. The process forthe preparation of a formulation as claimed in claim 14, wherein saidbhasmas are selected from a group consisting of Muktasukti bhasma, Lohabhasma, Abhraka bhasma, Swarnamaksika bhasma and Pravala bhasma.
 16. Theprocess for the preparation of a formulation as claimed in claim 14,wherein said finely powdered herbs comprises of finely powdered form of:Terminalia arjuna (dry stem bark), Sida rombifolia (dry root), Withaniasomnifera(dry root), Rubia cordifolia(dry root), Nardostachysjatamansi(dry root), Boerhavia diffusa(dry root), Picrorhiza kurroa(dryroot), Steriospermum suaveolens(dry root), Premna mucronata(dry root),Gmelina arborea(dry root), Aegle marmelos(dry root), Oroxylumindicum(dry root), Solanum indicum (dry root), Embelia ribes (dryfruit), Emblica officinalis (dry fruit), Terminalia chebula(dry fruit),Terminalia bellerica(dry fruit), Piper longum(dry fruit), Pipernigrum(dry fruit), Tribulus terrestris(dry fruit), Azhadirachta indica(dry bark), Solanum xanthocarpum (dry plant), Uraria picta(dry plant),Desmodium gangeticum (dry plant), Tinospora cordifolia (dry stem),Punica granatum (dry fruit pericarp), Zingiber officinalis (dryrhizome), Bamboo manna(exudate), Madhuka indica (dry flower), Holy basil(dry leaves) and Commiphora mukul (gum resin).
 17. The process for thepreparation of a formulation as claimed in claim 14, wherein saidgrinding decoction is a decoction of atleast one herb selected from thegroup consisting of Terminalia arjuna, Asparagus racemosus, Asafetida,Cuminum cyminum, Plumbago rosea, Baliospermum montanum, Ocimum sanctum,Aloevera, Plantago ovata, Steriospermum suaveolens, Premna mucronata,Gmelina arborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum,Uraria picta, Solanum indicum, Solanum xanthocarpum and Tribulusterrestris.
 18. A method of reducing the risks of Cardiovasculardiseases comprising, administering a therapeutically effective amount ofthe formulation claimed in claim
 1. 19. A method of treating andpreventing Cardiovascular diseases comprising, administering atherapeutically effective amount of the formulation claimed in claim 1.20. The method of treating and preventing Cardiovascular diseases asclaimed in claim 19, wherein said formulation is preferably administeredat a dose of two 500 mg tablets twice a day.
 21. A method of treatingand preventing Cardiovascular diseases comprising administering atherapeutically effective amount of the formulation claimed in claim 1.22. The method of treating and preventing Cardiovascular diseases asclaimed in claim 19, wherein said therapeutically effective amount is500 to 1000 mg administered one to three times a day.
 23. The method oftreating/preventing Cardiovascular diseases as claimed in claim 19,wherein said formulation is administered along with administration of atleast one other medication prescribed for treatment of Cardiovasculardiseases.